Abstract

Upon activation by the ligands Gas6 and Protein S, Tyro3/Axl/Mer (TAM) receptor tyrosine kinases promote phagocytic clearance of apoptotic cells and downregulate immune responses initiated by Toll-like receptors and type I interferons (IFNs). Many enveloped viruses display the phospholipid phosphatidylserine on their membranes, through which they bind Gas6 and Protein S and engage TAM receptors. We find that ligand-coated viruses activate TAM receptors on dendritic cells (DCs), dampen type I IFN signaling, and thereby evade host immunity and promote infection. Upon virus challenge, TAM-deficient DCs display type I IFN responses that are elevated in comparison to wild-type cells. As a consequence, TAM-deficient DCs are relatively resistant to infection by flaviviruses and pseudotyped retroviruses, but infection can be restored with neutralizing type I IFN antibodies. Correspondingly, a TAM kinase inhibitor antagonizes the infection of wild-type DCs. Thus, TAM receptors are engaged by viruses in order to attenuate type I IFN signaling and represent potential therapeutic targets.

(A) Left, WT BMDCs were incubated with increasing concentrations (0.5–2 nM) of purified human Protein S (Pros1), in either Gla-depleted cell culture medium alone (medium) or along with enveloped VSVg-pseudotyped HIV-1-derived virus that was produced in Gla-depleted medium (VSVg). Cell lysates were prepared for immunoblot 5 min after virus or Pros1 challenge. Mer was specifically immunoprecipitated from these samples, subjected to SDS-PAGE, and immunoblotted with a phosphotyrosine-specific antibody (IB: pY). Immunoblot analysis was used to confirm equal amounts of Mer in each sample (IB: Mer), and the amount of protein in each lysate was assessed by immunoblot for Gapdh (IB: Gapdh). Right, virus potentiation of Mer autophosphorylation in the presence of 1 nM Pros1 for 5 min is seen for the enveloped VSVg-pseudotyped virus, but not with WT MAV-1, a nonenveloped virus.(B) Left, the same experiments as in (A, left) except that WT BMDCs were incubated with 0.25–1 nM of recombinant mouse Gas6 (Gas6), and the tyrosine autophosphorylation of Axl was monitored (IB: pY). Right, Virus potentiation of Axl autophosphorylation in the presence of 1 nM Gas6 was seen only with the enveloped VSVg-pseudotyped virus and not with the nonenveloped MAV-1.(C) Microtiter wells, precoated with purified BSA, Gas6, or Pros1 (10 μg/ml ON for each protein) and incubated with equivalent amounts of VSVg-HIV (50 ng/ml p24 concentration) at 37°C for 2 hr with or without 5 mM EDTA, and the amount of bound virus was measured by ELISA for p24 protein (see Experimental Procedures). Error bars are SD of three samples. Results are representative of three independent experiments. See also .

(A–C) WT BMDCs (blue bars) or TAM TKO BMDCs (red bars) were challenged with the pseudotyped viruses of . Expression levels of IFNα4 (A), IFNβ (B), and SOCS1 (C) mRNAs were measured by qRT-PCR at the indicated time points after virus addition. The levels of the indicated mRNAs were normalized to control β-actin mRNA. *p < 0.05; **p < 0.01; ***p < 0.001. Results comparable to those for IFNα4 and IFNβ mRNAs were seen for measurements of TNFα, IRF-5, and IRF-7 mRNAs (see ). Error bars are SEM of samples from three independent experiments. See also .

The Primary Effect of TAM Receptor-Ligand Interactions on Virus Infection in BMDCs Is to Inhibit the Cellular Antiviral Response

(A) WT BMDCs and TAM TKO BMDCs were incubated with VSVg-pseudotyped virus in either the absence (−) or presence (+) of 100 μg each of neutralizing anti-mouse IFNα and IFNβ antibodies, and the levels of HIV-1 DNA were measured at 24 hr postinfection. Results were normalized to the level of viral DNA seen with WT BMDCs incubated with no antibody (100%, dashed blue line). ***p < 0.001. Error bars are SEM of samples from three independent experiments.(B) WT BMDCs were preincubated for 30 min with or without BMS-777607 and stimulated with Gas6 for 10 min. Receptor activation was monitored with the immunoprecipitation and immunoblotting protocol shown in ; this protocol employed a phosphotyrosine-specific antibody (pY). Immunoblot was used to confirm equal amounts of Mer and Axl in each sample.(C) WT BMDCs and TAM TKO BMDCs were incubated with EbGP-pseudotyped virus in the absence (−) or presence (+) of 300 nM BMS-777607, and the levels of HIV-1 viral DNA were measured at 24 hr postinfection. Results were normalized to those seen with WT BMDCs (100%, dashed blue line). **p < 0.01. Error bars are SEM of samples from three independent experiments.(D) WT BMDCs (blue bars), AM DKO BMDCs (red bars), and IFNAR KO BMDCs (green bars) were incubated with WNV in the absence (−) or presence (+) of 1 μM BMS-777607, and the levels of infectious virus (PFU per ml) were measured at 24 hr postinfection. ***p < 0.001. Error bars are SEM of three independent samples. Results are representative of three independent experiments. BMS-777607 effects are not due to cytotoxicity (see ).

A model showing that the major effect of TAM receptor-ligand interactions on enveloped virus infection of dendritic cells is at the level of inhibiting the cellular innate immune response, including TAM inhibition of type I IFN signaling.